There is presently a worldwide demand for an efficacious vaccine that reduces the risk of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) across cervicovaginal and rectal mucosae. In the female genital tract, it is thought that HIV-1 is initially “sampled” by motile intraepithelial or subepithelial dendritic cells and may initially infect mucosal T cells [Hussain et al., Immunology 85: 474-484 (9995); Parr et al., Biol. Reprod. 45: 261-265 (1991); Pope et al., J. Infect. Dis. 179: S427-S430 (1999); Spira et al., J. Exp. Med. 183: 215-225 (1996)]. In the rectum HIV-1 may enter via damaged epithelium or may cross an intact epithelial barrier via colonocytes or via specialized antigen transporting epithelial cells known as M cells [Amerongen et al., J. Acq. Immun Def. Synd. 4: 760-765 (1991); Bomsel, M., Nature Med. 3: 42-47 (1997)]. Once within the mucosa HIV-1 replicates in resident CD4+ T lymphocytes and/or macrophages and may be carried by these cells, as well as dendritic cells, to draining lymphoid organs within days after initial exposure [Ignatus et al., J. Med. Pathol. 27: 121-128 (1998); Miller et al., J. Med Primatol. 21: 64-68 (1992); Pope et al., Cell 78: 389-398 (1994); Stahl-Henning et al., Science 285: 1261-1265 (1999)].
Humoral Immunity:
Humoral immunity plays a critical role in preventing and/or modulating infection with the primate lentiviruses, including HIV, simian immunodeficiency virus (SIV), and the HIV-SIV chimeric virus SHIV [Moore & Burton, Nature Medicine 5: 142-144 (1999)]. For example, experiments in chimpanzees demonstrated that immunoglobulin (Ig) from the serum of HIV-infected individuals (HIVIG), monoclonal Ab (mAb), chimeric mAb, and anti-CD4-immunoglobulin IgG can all prevent infection with HIV; and that a human mAb to gp41 can significantly delay signs of infection [Prince et al., AIDS Res. Hum. Retrovir. 7: 971-973 (1991); Emini et al., Nature 355: 728-730 (1992); Emini et al., J. Virol. 64: 3674-3678 (1990); Conley et al., J. Virol. 70: 6751-6758 (1996)].
These studies of protection of chimpanzees by passive immunization suggest that the best correlates of immunoprophylaxis within in vivo studies are effective virus neutralizing activity in vitro and a slow Ab dissociation rate constant [Van Cott et al., J. Immunol. 153: 449-459 (1994)]. Similarly, most studies in mice reconstituted with human peripheral blood mononuclear cells exhibiting severe combined immunodeficiency syndrome (hu-PBL-SCID) have also demonstrated that pre- and postexposure protection against HIV infection can be mediated by murine mAb, human mAb, and mouse-human chimeric mAb [Safrit et al., AIDS 7: 17-21 (1992); Gauduin et al., J. Infect. Dis. 171: 1203-1209 (1995); Parren et al., AIDS 9: F1-F6 (1995); Gauduin et al., Nature Med. 3: 1389-1390 (1997)]. All of these studies suggest that Ab of appropriate specificities can prevent HIV and SIV infection with cell-free virions and of slowing viral replication and disease progression.
Active Immunization Studies:
Vaccine studies in primate models have increased our understanding of the interplay of viral replication and host immunity. Conjectured for a number of years, and now documented in several primate studies, is the observation that infection with live-attenuated viral vaccines induces strong cellular and humoral immunity, including neutralizing Ab effective against the macaque-grown challenge virus stocks, which can be considered primary isolates in this system.
The induction of these humoral responses is dependent upon a threshold of replication of the attenuated virus during primary viremia [Ruprecht et al., AIDS 10: S33-S40 (1996)]. Below this threshold, immune responses are weak and full protection is not seen except with very weak virus challenges; above the threshold, strong host immunity is observed in most animals and protection from infection with highly pathogenic SIV challenges ensues. These data and those obtained in vaccine studies with live-attenuated SIV, summarized by Table A below, support the notion that the level of attenuated virus replication during primary infection predicts whether the immune response is sufficient to block infection upon subsequent challenge with wild type virus.
Unfortunately, several examples of pathogenic effects from highly attenuated live viral vaccines were documented in five laboratories during the 1998 year, as summarized in a recent editorial [Cohen, J., Science 278: 24-25 (1997)]. Thus, it remains the difficult goal of vaccinologists either: (1) to construct live-attenuated viruses that are both effective and safe, or (2) to mimic the presentation of viral proteins observed in infection with recombinant antigens or with replicating or non-replicating vectors carrying appropriate genes or antigens.
TABLE A*Summary of representative HIV/SIV vaccine approaches in primate modelsNeutralizingRelative‘Sterilizing’ immunity (partial protectionantibodyCorrelate ofHost/viral pairpathogenicityor reduction in viral load)Challenge virusinducedprotectionChimpanzee/Lowgp120 subunit [Refs. 1 and 2]HIVSF2; HIVIIIB+YesHIV-1Vaccinia gp160 + V3 peptide [Ref. 3]HIVIIIB+YesDNA encoding gp160 [Ref. 4]HIVSF2+/−YesAdenovirus gp160 + gp120 subunit [Refs. 5 and 6]HIVSF2+, 1°YesCanarypox-gag-prot-env [Ref. 7]HIVIIIB+/−Yes(cell associated)Canarypox-gag-prot/env +/− subunit boost [Ref. 7]HIVDHI2−NoMacaque/Low(DNA encoding HIV gp120) [Ref. 8]SHIV−HXB2+YesSHIV(gp160 subunit) [Ref. 9]SHIV−HXB2+Yes(Vaccinia-gag-pol-env + gag-pol VLP, gp160 subunit)SHIV−HXB2+Yes[Ref. 9]gp120 subunit in ISCOMS + V2/V3 peptide boost [Ref. 10]SHIV-primary+YesLive-attenuated SIV; high replicative capacity [Refs. 11, 12 and 13]SHIV−HXB2/−NoSHIV−DHI2Vaccinia-gp160 + gp160 subunit [Ref. 9]SHIV−HXB2+YesDNA encoding gp160t + gp120 subunit [Ref. 14]SHIV−HXB2+YesMacaque/Moderate(gp160 subunit) [Ref. 15]SIVmneE11S+NoSIVmneE11s(Vaccinia-gp160 + gp160 subunit) [Ref. 15]SIVmneE11S;+Noor SIVmneSIVmne(uncloned)(Vaccinia-gag-pol + Gag-Pol VLP) [Ref. 15]SIVmneE11S+NoVaccinia-gag-pol-env − Gag-Pol-Env VLP [Ref. 15]SIVmneE11S+NoVaccinia-gag-pol; env + Gag-Pol VLP + gp160 subunit [Ref. 15]SIVmne+NoMacaque/High(Vaccinia-gp160 and gp160 subunit) S1Vmac251 [Ref. 15-18]SIVsmE660;+NoSIVsmE660SIVmac239or SIVmac251(Live-attenuated SIV; low replicative capacity) [Ref. 19]SIVmac251 (ivag)+Noor SIV239(Live-attenuated high-replicative capacity SHIV) [Refs. 12 and 20]SIVmac239 (ivag);−NoSIVsm (IR)(Adenovirus-gp120 + gp120 subunit) [Ref. 21]SIVmac251 (ivag)+YesLive-attenuated SIV; high replicative capacity [Refs. 22-25]SIVmac251+, 1°YesSIVsmmPRj6.6Yes, neutralization against homologous laboratory isolate; SIV, simian immunodeficiency virus; SHIV, simian-human immunodeficiency virus; 1°, includes neutralization of primary isolates.*Reproduced from Haigwood N. L. + S. Zolla-Pazner, AIDS 12: S121-S132 (1998), p. S127.ReferencesRef. 1: El-Amad et al., AIDS 9: 1313-1322 (1995).Ref. 2: Berman et al., Nature 345: 622-625 (1990).Ref. 3: Girard et al., Proc. Natl. Acad. Sci. USA 88: 542-546 (1991).Ref. 4: Boyer et al., Nature Med. 3: 526-532 (1997).Ref. 5: Lubeck et al., Nature Med. 3: 651-658 (1997).Ref. 6: Zolla-Pazner et al., J. Virol. 72: 1052-1059 (1998).Ref. 7: Girard et al., Virology 232: 98-104 (1997).Ref. 8: Robinson, H. L. AIDS 11: S109-S119 (1997).Ref. 9: Hu et al., 9th Annual Meeting of the National Cooperative Vaccine Development Group for AIDS, Bethesda, MD, 1997, Abstract No. 69.Ref. 10: Heeney et al., 15th Annual Symposium on Nonhuman Primate Models for AIDS, Seattle, WA, 1997, Abstract No. 5.Ref. 11: Dunn et al., AIDS Res. Hum. Retrovir. 13: 913-922 (1997).Ref. 12: Miller et al., J. Virol. 71: 1911-1921 (1997).Ref. 13: Shibata et al., J. Virol. 71: 8141-8148 (1997).Ref. 14: Letvin et al., Proc. Natl. Acad. Sci. USA 94: 9378-9383 (1997).Ref. 15: Hu et al., Immun. Lett. 51: 115-119 (1996).Ref. 16: Hirsch et al., J. Virol. 70: 3741-3752 (1996).Ref. 17: Ahmad et al., AIDS Res. Hum. Retrovir 10: 195-204 (1994).Ref. 18: Daniel et al., AIDS Res. Hum. Retrovir. 10: 839-851 (1994).Ref. 19: Marthas et al., J. Virol. 64: 3694-3700 (1990).Ref. 20: Quesda-Rolauder et al., AIDS Res. Hum. Retrovir. 12: 993-999 (1996).Ref. 21: Buge et al., J. Virol. 71: 8531-8541 (1997).Ref. 22: Wyand et al., J. Virol. 70: 3724-3733 (1996).Ref. 23: Lewis et al., 15th Annual Symposium on Nonhuman Primate Models for AIDS, Seattle, WA, 1997, Abstract No. 1.Ref. 24: Wyand et al., Nature Med. 3: 32-36 (1997).Ref. 25: Daniel et al., Science 228: 1201-1204 (1992).‘Prime/Boost’ and Subunit Vaccines Tested by Challenge with SHIV and SIV:
It has been shown that immunization with HIV-1LA1 gp160 vaccines, in a recombinant vaccinia virus priming and subunit boosting regimen, protected macaques against SIV HXBc2 challenge [Haigwood, N. L. and S. Zolla-Pazner, AIDS 12: S121-S132 (1998)]. Using the same challenge model, it was found subsequently that subunits alone were not protective (gp120; none out of three protected) or partially protective (gp160; two out of four protected). Complete protection was observed in all six macaques that received vaccinia virus-expressing HIV-1 gp160 and boosts of either gp120 (three out of three protected) or gp160 (three out of three protected). More complex immunogens including Env-bearing pseudovirion particles were partially effective in providing protection against SHIV challenge (three out of five protected). These data underline the importance of providing sufficient Env protein in vaccine preparations.
The HIV Envelope Glycoprotein:
An overview of the scientific reports shows that the envelope glycoprotein (env) of human immunodeficiency virus-1 (HIV-1) is synthesized as a precursor molecule gp160 and subsequently processed into its subunits gp120 and gp41. Gp120 is non-covalently associated with gp41 and contains the binding sites for CD4 molecules, i.e., the cellular receptors of HIV-1, and the chemokine receptors such as CCR4 and CXCR5. The gp41 subunit is anchored in the membrane and has a non-polar fusion peptide at its N-terminus. The gp120-gp41 molecule forms oligomers on the infected cell surface and on virions. Strong evidence for trimeric oligomers states has been reported at length in the published scientific literature.
The binding of gp120 to CD4 is thought to result in activation of the membrane fusion activity of gp41, leading to entry of the viral nucleocapsid into a cell. Evidence for a conformational change in the viral glycoprotein upon binding CD4 includes alterations in antibody reactivity, increased protease sensitivity and the dissociation of gp120.
Recent publications which factually support this summary overview include the following: Allan et al., Science 228: 1091-1094 (1985); Veronese et al., Science 229: 1402-1405 (1985); Dagleish et al., Nature 312: 763-767 (1984); Klatzman et al., Nature 312: 767-768 (1984); Madden et al., Cell 47: 333-348 (1986); Bosch et al., Science 244: 694-697 (1989); Kowalski et al., Science 237: 1351-1355 (1987); Gelderblom et al., Virology 156: 171-176 (1987); Pinter et al., Virology 83: 417-422 (1977); Schawaller et al., Virology 172: 367-369 (1989); Earl et al., J. Virol. 68: 3015-3026 (1994); Weiss et al., J. Virol. 64: 5674-5677 (1990); and Sattentau Q. and J. P. Moore, J. Exp. Med. 174: 407-415 (1991); Weissenhorn et al., PNAS 94: 6065-6069 (1997); Weissenhorn et al., EMBO J. 15: 1507-1514 (1997); Weissenhorn et al., Molecular Membrane Biologs 16: 3-9 (1998); and Weissenhorn et al., Nature 387: 426-430 (1997).
Antigen Structures which Induce Ab Responses:
Since the form of immunogen affects the type and specificity of the immune response, the nature of the immunogens found in natural infection that elicit Ab becomes a pivotal issue which impacts on vaccine design. Anti-HIV envelope polyclonal and monoclonal antibody preparations react with HIV-infected cells, implying that infected cells express envelope antigens that serve to both induce Ab and act as their targets. Thus, HIV+ sera and mAb to gp41 and the V3 and C5 regions of gp120 have been shown to stain cells infected with primary isolates and to mediate neutralization and/or Ab-dependent cell-mediated cytolysis (ADCC) [Zolla-Pazner et al., J. Virol. 69: 3807-3815 (1995); Tyler et al., J. Immunol. 145: 3276-3282 (1990); Alsmadi et al., J. Virol. 71: 925-933 (1997); Bauir et al., J. Immunol. 157: 2168-2173 (1996). This demonstrates that infected cells express virus-derived antigens. Oligomeric envelope proteins also are immunogenic.
As summarized in a recent paper [Haigwood, N. L. and S. Zolla-Pazner, AIDS 12: S121-S132 (1998)], while oligomer-specific mAb have only been described in immunized mice and rabbits, several human mAb have been described which show better reactivity with polymeric than with monomeric HIV envelope molecules. Amongst the first of these were human mAb to gp41 which preferentially react with oligomeric forms of gp41 on Western blot [Zolla-Pazner et al., N. Engl. J. Med. 320: 1280-1281 (1989); Pinter et al., J. Virol. 63: 2674-2679 (1989)]. Later studies suggested that mAb IgG1b12, specific for the CD4 binding domain preferentially binds to structures exposed on oligomeric envelope protein [Fouts et al., J. Virol. 71: 2779-2785 (1997)]; and mAb 2F5, specific for an epitope near the transmembrane region of gp41, binds to the oligomeric structure of gp41 in the virion envelope, resulting in neutralization [Muster et al., J. Virol. 68: 4031-4034 (1994)]. That all of these mAb also recognize structures on the monomeric forms of gp120 or gp41 is shown by the fact that the hybridoma cell lines producing these mAb were each selected using monomeric forms of these envelope glycoproteins.
Immune Responses to gp41:
Recently there has been a renewed interest in the immune response to gp41. The potential importance of Ab to gp41 is well demonstrated by the human mAb 2F5 which is specific for the ELDKW [SEQ ID NO:5]epitope near the transmembrane domain of gp41 and has broad neutralizing activity for laboratory-adapted strains and primary isolates of HIV [Muster et al., J. Virol. 68: 4031-40343 (1994)]. Other anti-gp41 mAb also have been shown to neutralize both laboratory-adapted and primary isolates of HIV [Hioe et al., Int. Immunol. 9: 1281-1290 (1997); Cotropia et al., AIDS Hum. Retrovir. 12: 221-232 (1996)]; and it was recently suggested that Ab to gp41 epitopes in the serum of HIV-infected individuals may play an important role in virus neutralization [McKeating et al., Virology 220: 450-460 (1996)].
Additional interest comes from research on the structure of gp41 and its role in infectivity. Thus, gp41, which mediates fusion between viral and cellular membranes, has been shown to consist of a rod-like molecule with a high alpha-helical content [Weissenhorn et al., EMBO J. 15: 1507-1514 (1996)]; and the structure of the fusogenic form appears to be composed of a six-helical bundle of two regions of the gp41 molecule. The core of the gp41 structure forms an extended, triple stranded coiled coil derived from a predicted leucine zipper domain approximately 30 residues from the N-terminal fusion peptide. A C-terminal a-helix packs in the reverse direction against the outside of the coiled coil following the groove between two core helices [Weissenhorn et al., Nature 387: 426-430 (1997); Chan et al., Cell 89: 263-273 (1997)]. The soluble forms of gp41 visualized by two crystal structures contain gp41 residues 30 to 79 and 113 to 153 [Weissenhorn et al., Nature 387: 426-430 (1997)] and a smaller construct contains residues 35 to 70 and 117 to 150 [Chan et al., Cell 89: 263-273 (1997)]. The conformational and linear epitopes exposed on gp41 appear to be different in gp41/gp120 nonfusogenic configuration and in the fusion active conformation [Sattentau et al., 1995; Weissenhorn et al., EMBO J. 15: 1507-1514 (1996)].
It has been suggested that the conformational structure of gp41 provides the fusion-active capability for gp41. A general model was presented where the complex of gp120/gp41 undergoes major conformational changes after interaction with cellular receptors CD4 and chemokine receptors [Berger et al., Annu Rev Immunol 17: 886-900 (1999)]. The conformational changes occurring in gp41 are thought to open up intermediary conformational states and the complete refolding of the molecule results in the helical hairpin structure observed by crystallography. This process is thought to pull two membranes into close proximity and induce fusion of viral and cellular membranes [see FIG. 3 in Weissenhorn et al., Nature 387: 426-430 (1970]. It is conceivable that monoclonal antibodies that either block the formation of the helical hairpin, like gp41 specific peptides [Kilby et al., Nat. Med. 4: 1302-1307], or block the aggregation of gp41 helical hairpin structures (a number of trimers are necessary at the site of fusion [Danilei et al., J. Cell Biol. 133: 559-569 (1996)]) at the site of fusion may inhibit membrane fusion and thus infection.
HIV Envelope Glycoprotein Variants, Synthetic Chimeras, and gp41 Structure:
In recognition of the fact that the HIV envelope subunit gp41 plays such a critical role in viral entry by initiating fusion of viral and cellular membranes, Weissenhorn and colleagues have synthesized new construct variants of the ectodomain of HIV-1 and the env gp41 subunit in particular. Thus it has been shown that the env subunit gp41 forms a slightly soluble, (alpha)-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide [Weissenhorn et al., EMBO J. 15: 1507-1514 (1996)]; and also that a rod shaped chimera of a trimeric GCNA zipper and the HIV-1 gp41 ectodomain can be synthesized and expressed in E. coli and solubilized by proteolysis [Weissenhorn et al., Proc. Natl. Acad. Sci. USA 94: 6065-6069 (1997)]; and that the atomic structure of the ectodomain from HIV gp41 is an extended, triple stranded alpha-helical coil with the N-terminus at its tip [Weissenhorn et al., Nature 387: 426-430 (1997)]. The core of the molecule forms an extended, triple-stranded alpha-helical coiled coil with the N-terminus at its tip. A C-terminal alpha-helix packs in the reverse direction against the outside of the coiled coil following the groove between two core helices. This arrangement places the N-terminal fusion peptide and the C-terminal transmembrane region at the same end of the rod-shaped molecule [Weissenhorn et al., 1997].
These reported investigations and published papers centered in particular upon finding new synthetic chimeras which might substantially increase the solubility of gp41—and thus possibly increase the number of epitopes exposed as well as the potential antigenicity of the gp41 amino acid sequences. As noted in these recently published papers, the crystal structures were derived from different sources. Core fragments of gp41 were either assembled from synthetic peptides [Chan et al., 1997], or expressed in E. coli and solubilized with a trimeric GCN4 zipper fused to the predicted N-terminal coiled coil and trimmed by proteolysis [Weissenhorn et al., 1997]. Alternatively, E. coli expressed N-terminal and C-terminal helical regions were connected by a synthetic linker [Tan et al., 1997].
All three gp41 structures constructed in this manner (as described in the published papers) are missing the N-terminal region containing the hydrophobic fusion peptide and the loop that connects a N-terminal core helix with a C-terminal helix. The HIV gp41/GCN4 chimera is missing 39 linker residues, which would contain a short disulphide linked loop and two carbohydrate sites [Weissenhorn et al., Nature (1997)]. Although the disulphide linked loop C-terminal of the coiled coil region is characteristic for all retroviral and filoviral fusion proteins, its function is not yet known. The disulphide linked loop in HIV might play a role in the change of conformation as determined by differential antibody reactivity [Weissenhorn et al., EMBO J. (1996)].
Gp41 sequences of different HIV subtypes show a remarkable conservation for the N-terminal coiled coil as well as for the C-terminal residues that interact with the N-terminal core structure [Weissenhorn et al., Nature (1997); Chan et al., Cell (1997)]. Indeed, there are only conservative changes within interfaces of two N-terminal helices and one C-terminal helix, and most of the differences are on the outside of the C-terminal helix, exposed to the solvent. This reveals that the C-terminal helix packs into a highly conserved groove along the core coiled coil, which is remarkable considering the sequence variability in HIV [Myers et al., 1995].
In addition, there are several lines of evidence that the gp41 membrane fusion protein exists in two conformations: a native conformation in complex with gp120; and a fusion-conformation. First, receptor binding was shown to increase the exposure of gp41 epitopes [Sattentau and Moore, 1992] as well as to stimulate the dissociation of gp120 from gp41 [Kirsh et al., 1990; Moore et al., 1990; Hart et al., 1991]. Antibodies raised against native gp41 (in complex with gp120) [Earl et al., 1994] showed a differential reactivity with gp41 expressed (without gp120) in insect cells. Some of the antibodies were mapped to the short disulphide linked loop and recognized native gp41 but not the fusion conformation [Weissenhorn et al., 1996].
Second, direct evidence arises from a number of mutagenesis studies, which showed that residue changes especially within the heptad positions of the central coiled coil affect infectivity and membrane fusion, but not processing and cell surface expression of gp41/gp120 complexes [Dubay et al., 1992; Cao et al., 1993; Chen et al., 1993; Chen 1994]. This indicates that these changes are tolerated in the native conformation but not in the fusion conformation.
Third, peptides derived from the gp41 sequence, like DP-107 (part of the N-terminal coiled coil) and DP-178 (C-terminal helix, with an expression towards the transmembrane region), have potent anti-viral activity [Jiang et al., 1992; Wild et al., 1992; 1994; Lawless et al., 1996]. The structure of gp41 confirms the view that these derived peptides expert their effect by interacting with gp41 during the receptor induced conformational change. This is also consistent with the finding that the assembled complex (N- and C-terminal helices) has no anti-viral activity [Lu et al., 1995]. The conformation of gp41, as observed in the crystal structure, shows a temperature dependent denaturation at approximately 80° C. [Blacklow et al., 1995; Lu et al., 1995; Weissenhorn et al., 1996]; which makes it unlikely that the complex comes apart and interacts with individual peptides. Kinetic measurements of receptor-activated conformational changes showed that these changes are initiated within a few minutes and completed after 20 min [Jones et al., 1998]. It is also remarkable that the C-terminal peptide (DP-107) remains active even when added after mixing of the target cells [Munoz-Barroso et al., 1998]. The C-terminal peptide DP-178 does not interact with native gp41, but binds to gp41 after induction of receptor mediated conformational changes, an event which confirms the structural changes in gp41 upon receptor binding [Furuta et al., 1998].
Immunization:
It is generally agreed that multiple immune effectors participate in prevention, containment and clearance of HIV infection. To prevent infection of host target cells, antibodies are required. After the first target cells have been infected with virus, it is important to have cytotoxic T lymphocytes (CTLs) as well as antibodies to reduce cell-to-cell spread and kill infected cells. The exact amounts of specific antibodies or CTLs required for mucosal or systemic protection against HIV are not known. However, it seems clear that an effective HIV vaccine should evoke antibodies that can bind to virus and prevent attachment of virus to target cells, as well as CTLs that can eliminate any cells that become infected.
If virus is transmitted directly into the body as through injection, accidental needle stick or damaged skin or mucosa, then antibodies and CTLs in the bloodstream, both of which can readily enter tissues, are most important for protection. Vaccines that are injected intramuscularly or intradermally are generally most efficient for inducing these immune effectors in the blood. However, a large proportion of HIV infections are the result of mucosal transmission. This most often occurs via the cervical-vaginal mucosa and the rectal mucosa, but may also occur via the oral mucosa and nasopharyngeal mucosa. The extent to which antibodies and CTLs from blood can prevent, contain or clear mucosal infections at a very early stage, before virus has spread systemically, is not yet clear. Mucosal surfaces have an additional immune protection mechanism: transport of antibodies into secretions. Secretory antibodies can provide the first line of defense, preventing contact of viruses with the mucosal surface and thereby preventing entry into the body and target cell infection altogether (see below). Secretory antibodies are generally not induced by systemic immunization. Immunization via mucosal surfaces is usually required to evoke secretory antibodies and local CTLs and antibodies in mucosal tissues. In experimental animals and humans, these effectors are induced most efficiently at the mucosal site where the vaccine was administered [Haneberg et al., Infect. Immun. 62: 15-23 (1994); Kozlowski et al., Infect. Immun. 65: 1387-1394 (1997)]. In addition, vaccines administered mucosally may induce antibodies in the bloodstream.
The exact composition of an optimal HIV vaccine, or the protocols or routes by which it should be administered, are not yet established. One type of protocol currently being tested is a combination prime-boost approach in which a live vaccine vector (such as fowlpox) carrying HIV genes is given by injection to prime the immune system, followed by booster doses consisting of subunit antigens (usually the HIV envelope proteins gp120 or gp160). The subunit boost appears to be essential for induction of immune responses in serum. As expected, mucosal secretory antibodies have not been detected in animal experiments and human trials using such protocols. Alternative protocols for induction of secretory antibodies are currently being considered. For example, one possibility is administration by injection of a prime consisting of live HIV vaccine vector or DNA encoding HIV antigens, followed by boosts consisting of HIV envelope antigens, administered via a mucosal surface. The exact form or composition of envelope antigens most appropriate for mucosal administration are not yet established.
Secretory IgA Antibodies:
There is mounting epidemiological and experimental evidence that the presence of secretory immunoglobulin A (S-IgA) antibodies directed against the HIV envelope protein gp41 may reduce or prevent sexual transmission of HIV-1 [Lehner et al., Nature Med. 2: 767-775 (1996)]. For example, studies in Kenya and Thailand demonstrated a positive correlation in female sex workers between resistance to HIV-1 infection and the presence of anti-gp160 S-IgA antibodies in cervico-vaginal secretions [Beyer et al., J. Infect. Dis. 179: 59-67 (1999); Kaul et al., AIDS 13: 23-29 (1999)]. A similar correlation was observed in HIV-seronegative women with HIV-seropositive partners in Italy [Mazzoli et al., Nature Med. 3: 1250-1257 (1997)]. IgA isolated from secretions of exposed-uninfected women in both Kenya and Italy inhibited transcytosis of HIV across cultured epithelial monolayers in vitro [Devito et al., J. Immunol. 165: 5170-5176 (2000)]. However, Beyrer et al. [J. Inf. Dis. 179: 59-67 (1999)] found that anti-gp160 IgA antibodies in cervico-vaginal secretions of HIV-resistant sex workers failed to react with gp120, suggesting the antibodies may recognize epitopes located on gp41. Indeed, a recent study has mapped the epitopes recognized by anti-gp160 S-IgA antibodies from cervico-vaginal secretions of exposed-serononegative sex workers to amino acids 65-68 (LQAR) of the gp41 ectodomain [Pastori et al., J. Biol. Regul. Homeo. Agts. 14: 15-21 (2000)]. In vitro, anti-gp41 IgA antibodies purified from colostra of HIV-infected women prevented viral transmission across intestinal epithelial cell monolayers [Bomsel et al., Immunity 9: 277-287 (1998)].
Thus, an important goal of an effective HIV vaccine strategy should be to induce anti-gp41 antibodies in secretions of uninfected individuals. However, only two reports have examined the mucosal immunogenicity in mice of peptides representing epitopes of gp41 expressed via live recombinant viral vectors [Durrani et al., J. Immunol. Meth. 220: 93-103 (1998); Muster et al., J. Virol. 69: 6678-6686 (1995)]. Nevertheless, some additional epitopes that might be useful for mucosal protection immunologically are present in the gp41 ectodomain.
The Continuing Problems:
Induction of antigen-specific IgA on mucosal surfaces poses several challenges. First, mucosal delivery of antigens is required because S-IgA antibodies are induced after mucosal but not parenteral immunization [Mestecky et al., FEMS Imm. Med. Micro. 27: 351-355 (2000)]. Vaccines taken up at mucosal sites evoke proliferation of IgA-committed, antigen-sensitized lymphoblasts in organized mucosa-associated lymphoid tissue (O-MALT) that eventually seed local and distant mucosal and glandular tissues with IgA-producing plasma cells [Brandtzaeg et al., in Mucosal Immunology, Acad. Press, 1999, pp. 439-468]. Intranasal immunization of humans, for example, can lead to the appearance of antigen-specific IgA in the secretions of the airways, small intestine, rectum, and female genital tract [Bergquist et al., Infect. Imm. 65: 2676-2684 (1997); Kozlowski et al., Immunol. Lett. 69: 98 [Abst. 23.8] (1999)]. However, one major recognized difficulty in mucosal immunization is that many antigens fail to cross epithelial barriers and gain access to the O-MALT. A second major problem is that large doses of protein antigen are typically required to achieve sufficient sampling by the MALT due to the presence of mucus, proteases and natural clearance mechanisms on mucosal surfaces [McGhee et al., in Mucosal Immunology, Acad. Press, 1999, pp. 741-757]. A third major difficulty is the current absence of identifiable antigens that can be sampled by the MALT after mucosal immunization and evoke anti-gp41 S-IgA antibodies that recognize clinically relevant isolates of HIV-1.